Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment.

Lineage commitment during embryonic stem cell (ESC) differentiation is controlled not only by a gamut of transcription factors but also by epigenetic events, mainly histone deacetylation and promoter DNA methylation. The DNA demethylation agent 5'-aza-2'-deoxycytidine (AzadC) has been widely described as an effective promoter of cardiomyogenic differentiation in various stem cell types. However, its toxicity and instability complicate its use. Therefore, the purpose of this study was to examine the effects of zebularine (1-(ß-D-ribofuranosyl)-1,2-dihydropyrimidin-2-1), a stable and non-toxic DNA cytosine methylation inhibitor, on mouse ESC (mESC) differentiation. Herein, we report that treating embryoid bodies, generated from mESCs, with 30 µM zebularine for 7 days led to greater cell differentiation and induced the expression of several cardiac-specific markers that were detected using reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, immunostaining and flow cytometry. Zebularine enhanced the expression of cardiac markers and the appearance of beating cells that responded to cardiac drugs, including ion channel blockers (diltiazem) and ß-adrenergic stimulators (isoproterenol). Gene promoter methylation status was assessed using methylation-specific PCR (MSP) and validated by bisulfite sequencing analysis. Global gene expression profiling using microarrays showed that zebularine-differentiated cells are distinct from control ESCs. Pathway analysis revealed an enhancement of cellular processes such as embryonic development, cardiovascular system development and function. In addition, the whole-cell proteins exhibited different profiles as analyzed by two-dimensional differential-in-gel-electrophoresis. Our results indicate that zebularine regulates mesodermal differentiation of mESCs, controls promoter methylation of crucial cardiac genes and may help to improve cardiomyogenic differentiation.

Nitric oxide repression of Nanog promotes mouse embryonic stem cell differentiation.

Exposure of mouse embryonic stem (mES) cells to high concentrations of chemical nitric oxide (NO) donors promotes differentiation, but the mechanisms involved in this process at the gene expression level are poorly defined. In this study we report that culture of mES cells in the presence of 0.25-1.0 mM diethylenetriamine nitric oxide adduct (DETA-NO) leads to downregulation of Nanog and Oct4, the two master genes involved in the control of the pluripotent state. This action of NO was also apparent in the human ES cell line, HS 181. The suppressive action of NO on Nanog gene depends on the activation of p53 repressor protein by covalent modifications, such as pSer15, pSer315, pSer392 and acetyl Lys 379. NO-induced repression of Nanog is also associated with binding of trimethylated histone H3 and pSer315 p53 to its promoter region. In addition, exposure to 0.5 mM DETA-NO induces early differentiation events of cells with acquisition of epithelial morphology and expression of markers of definitive endoderm, such as FoxA2, Gata4, Hfn1-beta and Sox 17. This phenotype was increased when cells were treated with valproic acid (VPA) for 10 days.

Low concentrations of nitric oxide delay the differentiation of embryonic stem cells and promote their survival.

Nitric oxide (NO) is an intracellular messenger in several cell systems, but its contribution to embryonic stem cell (ESC) biology has not been characterized. Exposure of ESCs to low concentrations (2-20 µM) of the NO donor diethylenetriamine NO adduct confers protection from apoptosis elicited by leukaemia inhibitory factor (LIF) withdrawal. NO blocked caspase 3 activation, PARP degradation, downregulation of the pro-apoptotic genes Casp7, Casp9, Bax and Bak1 and upregulation of the anti-apoptotic genes Bcl-2 111, Bcl-2 and Birc6. These effects were also observed in cells overexpressing eNOS. Exposure of LIF-deprived mESCs to low NO prevented the loss of expression of self-renewal genes (Oct4, Nanog and Sox2) and the SSEA marker. Moreover, NO blocked the differentiation process promoted by the absence of LIF and bFGF in mouse and human ESCs. NO treatment decreased the expression of differentiation markers, such as Brachyury, Gata6 and Gata4. Constitutive overexpression of eNOS in cells exposed to LIF deprivation maintained the expression of self-renewal markers, whereas the differentiation genes were repressed. These effects were reversed by addition of the NOS inhibitor L-NMMA. Altogether, the data suggest that low NO has a role in the regulation of ESC differentiation by delaying the entry into differentiation, arresting the loss of self-renewal markers and promoting cell survival by inhibiting apoptosis.

Cell therapy for diabetes mellitus: an opportunity for stem cells?

Diabetes is a chronic disease characterized by a deficit in beta cell mass and a failure of glucose homeostasis. Both circumstances result in a variety of severe complications and an overall shortened life expectancy. Thus, diabetes represents an attractive candidate for cell therapy. Reversal of diabetes can be achieved through pancreas and islet transplantation, but shortage of donor organs has prompted an intensive search for alternative sources of beta cells. This achievement has stimulated the search for appropriate stem cell sources. Both embryonic and adult stem cells have been used to generate surrogate beta cells or otherwise restore beta cell functioning. In this regard, several studies have reported the generation of insulin-secreting cells from embryonic and adult stem cells that normalized blood glucose values when transplanted into diabetic animal models. Due to beta cell complexity, insulin-producing cells generated from stem cells do not possess all beta cell attributes. This indicates the need for further development of methods for differentiation and selection of completely functional beta cells. While these problems are overcome, diabetic patients may benefit from therapeutic strategies based on autologous stem cell therapies addressing late diabetic complications. In this article, we discuss the recent progress in the generation of insulin-producing cells from embryonic and adult stem cells, together with the challenges for the clinical use of diabetes stem cell therapy.

Macrophage inducible nitric oxide synthase gene expression is blocked by a benzothiophene derivative with anti-HIV properties.

Nitric oxide (NO) has been shown to mediate multiple physiological and toxicological functions. The inducible nitric oxide synthase (iNOS) is responsible for the high output generation of NO by macrophages following their stimulation by cytokines or bacterial antigens. The inhibition of TNF alpha-stimulated HIV expression and the anti-inflammatory property of PD144795, a new benzothiophene derivative, have been recently described. We have now analyzed whether some of these properties could be mediated by an effect of PD144795 on NO-dependent inflammatory events. We show that PD144795 suppresses the lipopolysaccharide-elicited production of nitrite (NO(-)(2)) by primary peritoneal mouse macrophages and by a macrophage-derived cell line, RAW 264.7. This effect was dependent on the dose and timing of addition of PD144795 to the cells. Suppression of NO(-)(2) production was associated with a decrease in the amount of iNOS protein, iNOS enzyme activity and mRNA expression. The effect of PD144795 was partially abolished by coincubation of the cells with LPS and IFN gamma. However, the inhibitory effect of PD144795 was not abrogated by the simultaneous addition of LPS and TNF alpha, which indirectly suggests that the effect of PD144795 was not due to the inhibition of TNF alpha synthesis. Additionally, PD144795 did not block NF-kappa B nuclear translocation induced by LPS. Inhibition of iNOS gene expression represents a novel mechanism of PD144795 action that underlines the anti-inflammatory effects of this immunosuppressive drug.

Evidence for involvement of c-Src in the anti-apoptotic action of nitric oxide in serum-deprived RINm5F cells.

The mechanism by which nitric oxide (NO) protects from apoptosis is a matter of debate. We have shown previously that phosphorylation of tyrosine residues participates in the protection from apoptosis in insulin-producing RINm5F cells (Inorg. Chem. Commun. 3 (2000) 32). Since NO has been reported to activate the tyrosine kinase c-Src and this kinase is involved in the activation of protein kinase G (PKG) in some cell systems, we aimed at studying the contribution of c-Src and PKG systems in anti-apoptotic actions of NO in serum-deprived RINm5F cells. Here we report that exposure of serum-deprived cells to 10 microM DETA/NO results in protection from degradation of the anti-apoptotic protein Bcl-2, together with a reduction of cytochrome c release from mitochondria and caspase-3 inhibition. Studies with the inhibitors ODQ and KT-5823 revealed that these actions are dependent on both activation of guanylate cyclase and PKG. DETA/NO was also able to induce autophosphorylation and activation c-Src protein both in vivo and in vitro and active c-Src was able to induce tyrosine phosphorylation of Bcl-2 in vitro. The c-Src kinase inhibitor PP1 abrogated the actions of DETA/NO on cGMP formation, PKG activation, caspase activation, cytochrome c release from mitochondria, and Bcl-2 phosphorylation and degradation in serum-deprived cells. We thus propose that activation of c-Src is an early step in the chain of events that signal cGMP-dependent anti-apoptotic actions of NO in mitocohondria.

Sodium nitroprusside-induced mitochondrial apoptotic events in insulin-secreting RINm5F cells are associated with MAP kinases activation.

Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA-NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K gamma gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kgamma. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.

Methylation-dependent gene silencing induced by interleukin 1beta via nitric oxide production.

Interleukin (IL)-1beta is a pleiotropic cytokine implicated in a variety of activities, including damage of insulin-producing cells, brain injury, or neuromodulatory responses. Many of these effects are mediated by nitric oxide (NO) produced by the induction of NO synthase (iNOS) expression. We report here that IL-1beta provokes a marked repression of genes, such as fragile X mental retardation 1 (FMR1) and hypoxanthine phosphoribosyltransferase (HPRT), having a CpG island in their promoter region. This effect can be fully prevented by iNOS inhibitors and is dependent on DNA methylation. NO donors also cause FMR1 and HPRT gene silencing. NO-induced methylation of FMR1 CpG island can be reverted by demethylating agents which, in turn, produce the recovery of gene expression. The effects of IL-1beta and NO appear to be exerted through activation of DNA methyltransferase (DNA MeTase). Although exposure of the cells to NO does not increase DNA MeTase gene expression, the activity of the enzyme selectively increases when NO is applied directly on a nuclear protein extract. These findings reveal a previously unknown effect of IL-1beta and NO on gene expression, and demonstrate a novel pathway for gene silencing based on activation of DNA MeTase by NO and acute modification of CpG island methylation.

Phenylarsine oxide increases intracellular calcium mobility and inhibits Ca(2+)-dependent ATPase activity in thymocytes.

A rise in intracellular Ca(2+) levels has been implicated as a regulatory signal for the initiation of lymphocyte proliferation. In the present study the mechanism underlying the elevation of [Ca(2+)] induced by phenylarsine oxide [PAO] was investigated in thymocytes. This agent inhibits HIV-1 replication and also NF-kappaB-mediated activation. It has been reported that the PAO-induced Ca(2+) elevation results from an enhanced plasma membrane calcium permeability in T cells. Here, we present biochemical evidence that the PAO-induced Ca(2+) increase was independent of external Ca(2+). Consistent with these facts, when [Ca(2+)](i) was depleted by prolonged incubation of the cells in Ca(2+)-free medium, PAO addition did not lead to [Ca(2+)](i) increase. These data indicate the involvement of intracellular organelles of thymocytes as the source of Ca(2+). Moreover, evidence is presented that PAO inhibited Ca(2+)-dependent ATPase activity from thymocytes and sarcoplasmic reticulum from skeletal muscle. This inhibition was dose-dependent, with a IC(50) of about 30 microM for both preparations of enzyme. The ability of PAO to inhibit Ca(2+)-dependent ATPase represents a novel mechanism of action for this drug. Present data suggest that the PAO-dependent [Ca(2+)](i) increase could be mainly the result of inhibition of Ca(2+)-dependent ATPase. In addition, we describe also a Ca(2+)-dependence for PAO effect on tyrosine phosphorylation.

NO induces a cGMP-independent release of cytochrome c from mitochondria which precedes caspase 3 activation in insulin producing RINm5F cells.

Exposure of RINm5F cells to interleukin-1beta and to several chemical NO donors such as sodium nitroprusside (SNP), SIN-1 and SNAP induce apoptotic events such as the release of cytochrome c from mitochondria, caspase 3 activation, Bcl-2 downregulation and DNA fragmentation. SNP exposure led to transient activation of soluble guanylate cyclase (sGC) and prolonged protein kinase G (PKG) activation but apoptotic events were not attenuated by inhibition of the sGC/PKG pathway. Prolonged activation of the cGMP pathway by exposing cells to the dibutyryl analogue of cGMP for 12 h induced both apoptosis and necrosis, a response that was abolished by the PKG inhibitor KT5823. These results suggest that NO-induced apoptosis in the pancreatic beta-cell line is independent of acute activation of the cGMP pathway.

Oxidative stress triggers STAT3 tyrosine phosphorylation and nuclear translocation in human lymphocytes.

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ ions, that.OH generated from H2O2 through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

Maternal serum nitric oxide levels associated with biochemical and clinical parameters in hypertension in pregnancy.

OBJECTIVES: To measure maternal serum concentrations of total nitrites, as an index of nitric oxide synthesis, in normal and hypertensive pregnant women, and to examine the correlation between these concentrations and several variables of clinical interest. STUDY DESIGN: A total of 60 women in four different groups were studied: 10 normotensive pregnant women, 17 pregnant women with preeclampsia, 18 pregnant women with gestational hypertension and 15 pregnant women with chronic hypertension. Serum nitrite levels were determined using the Griess reaction after reduction with nitrate reductase. RESULTS: Serum nitrite levels were higher in preeclamptic women (34.11+/-14 micromol/l, P=0.04), lower in chronic hypertensive women (19.56+/-6.46 micromol/l, P=0.04) and similar in women with gestational hypertension (26.97+/-9.44 micromol/l) in comparison to the control group (25.37+/-7.24 micromol/l). Serum nitrite levels in preeclamptic women had significant positive correlations with hematocrit, fasting insulinemia, and apolipoprotein B and negative correlations with platelet count, serum phosphorus and glucose:insulin ratio. In pregnant women with chronic hypertension a negative correlation was found between serum nitrite levels and active partial thromboplastin time. In pregnant women with gestational hypertension, serum nitrite levels had negative correlations with birthweight and 24-h urine calcium, and positive correlations with mean corspuscular hemoglobin, 24-h urine sodium and maternal age. CONCLUSIONS: We suggest that in women with preeclampsia, a higher maternal nitric oxide level may act as a compensatory mechanism against hemoconcentration and platelet aggregation and that nitric oxide production may be related to some metabolic events. In women with gestational hypertension, higher serum nitrite levels may be related to clinical and biochemical findings common in preeclampsia. In chronic hypertension, a lower maternal nitric oxide level is related to the status of coagulation.

Inhibitory effect of albumin-derived advanced glycosylation products on PMA-induced superoxide anion production by rat macrophages.

Advanced glycosylation end products (AGE) are implicated in many of the complications of diabetes. In the same way, infectious diseases are frequently associated with this disease. An impaired respiratory burst in macrophages may be a cause of infectious complications in diabetic patients. To establish a possible mechanism of this altered cell function, we have analyzed the effect of AGE-modified proteins on PMA-dependent superoxide anion production (O2.-) from normal rat peritoneal macrophages. We have used AGE-modified bovine serum albumin (AGE-BSA) prepared by incubation with glucose. AGE-BSA partially inhibits the phorbol ester-dependent superoxide production by macrophages in vitro. The specificity of this inhibitory effect is demonstrated by the fact that aminoguanidine, an inhibitor of the formation of AGE products, fully prevents the effect of AGE-BSA in vitro. Macrophages from diabetic rats shown an inhibition on PMA dependent-O2.- production. However, the treatment in vivo with aminoguanidine produced a cancelation of the inhibitory effect observed in the diabetic state. These data suggest that AGE-modified proteins could be implicated in the impairment of macrophage respiratory burst in diabetes.

Nicotinamide inhibits inducible nitric oxide synthase enzyme activity in macrophages by allowing nitric oxide to inhibit its own formation.

Nitric oxide (NO) production by macrophages is mainly regulated by induction of nitric oxide synthase (iNOS) by cytokines and microbial products. Nicotinamide (NIC) inhibits NO production by activated macrophages in a dose dependent manner. NIC also inhibits NOS enzyme activity in extracts from activated macrophages. The inhibition was noncompetitive with L-arginine (Ki 13.37 +/- 4.40 mM, n=3), uncompetitive versus NADPH (Ki 3.06 +/- 0.17 mM, n=3) and tetrahydrobiopterin. Finally, the inhibition by nicotinamide was fully reversed by scavenging NO with hemoglobin. We suggest that NIC acts by allowing NO to inhibit its own formation.

A newly synthesized molecule derived from ruthenium cation, with antitumour activity, activates NADPH oxidase in human neutrophils.

To determine the nature of the mechanism by which certain derived ruthenium (Ru) complexes induce regression in tumour growth, we have investigated the possibility that this mechanism was associated with an increase of superoxide anion (O2-. production by phagocytic cells, which are usually found in tumour nodes. Here we present evidence that a newly synthesized complex, Ru3+-propylene-1, 2-diaminotetra-acetic acid (Ru-PDTA), derived from Ru and the sequestering ligand (PDTA), specifically stimulates O2-. production. This increase was associated with the translocation of cytosolic factors p47(phox) and p67(phox) of NADPH oxidase to the plasma membrane. The Ru-PDTA-complex-dependent O2-. production was abrogated by staurosporine, partially inhibited by diphenylene iodonium, and it was insensitive to pertussis toxin or dibutyryl cyclic AMP pretreatment. An increase of cytosolic Ca2+ levels were also detected in neutrophils treated with the Ru-PDTA complex. Also, Ru-PDTA complex induced the phosphorylation of tyrosine residues of several proteins as assessed by Western blotting. Present data are consistent with the possibility that Ru-PDTA-dependent antitumour effects are due in part to the complex's ability to stimulate the release of toxic oxygen metabolites from phagocytic cells infiltrating tumour masses.

Protection from nicotinamide inhibition of interleukin-1 beta-induced RIN cell nitric oxide formation is associated with induction of MnSOD enzyme activity.

We have studied the long-term effects of nicotinamide (NIC) on the synthesis of NO by insulin producing cells. NIC delays the formation of nitrite by interleukin (IL)-1 beta-(IL-1, 25 U/ml)-stimulated RINm5F cells, and previous exposure of cells to IL-1 for 15 h prevents this effect. The delay is associated with a lack of cytokine-induced inducible nitric oxide synthase (iNOS) enzyme activity in cell extracts. NIC (20 mM) inhibits NO synthase (NOS) activity in extracts from cells incubated with IL-1 for 6 h and 24 h, and oxyhemoglobin counteracts this inhibition. Hence, NIC could scavenge O2- and allow NO to inhibit the enzyme. The NO donor SIN-1 inhibits in a concentration-dependent manner iNOS activity, and the effect is potentiated by NIC. In intact cells, protection from NIC is associated with IL-1-induced expression of MnSOD activity, and reversible blockade of iNOS expression with pyrrolidine dithiocarbamate counteracts the NIC effect. We conclude that O2- plays a role in preventing NO inhibition of iNOS. The loss of this action coincides with the induction of MnSOD enzyme activity. In addition, the stimulation by NIC of IL-1-induced nitrite production in pyrrolidine dithiocarbamate-treated cells is a novel action that should be considered when the drug is proposed as potential agent for the prevention of insulin-dependent diabetes mellitus.

N-monomethyl-arginine and nicotinamide prevent streptozotocin-induced double strand DNA break formation in pancreatic rat islets.

The impact of short term in vitro exposure to the diabetogenic drug streptozotocin on pancreatic islet glucose metabolism, insulin secretion, DNA fragmentation and cell viability, was studied. Streptozotocin impaired cell viability as well as insulin secretion and the oxidation of glucose. These effects were partially counteracted by inhibition of the inducible form of nitric oxide synthase with N-monomethyl-arginine and by scavenging oxygen free radicals with nicotinamide. Isolated islets underwent double strand DNA fragmentation after 24 h in culture. The degree of DNA breakdown was strongly enhanced by exposure of the islets to 0.55 mM streptozotocin for 30 min before culture. Prevention of streptozotocin-induced cleavage of islet DNA was obtained with N-monomethyl-arginine and nicotinamide. These data suggest that the generation of reactive oxygen and nitrogen species is involved in the deleterious action of streptozotocin on pancreatic islet tissue. A role for oxygen radicals generated during streptozotocin-induced islet cell damage as possible mediators of the expression of the inducible form of nitric oxide synthase and the scavenging action of nicotinamide on these radicals, is then proposed.

Expression of the citrulline-nitric oxide cycle in rodent and human pancreatic beta-cells: induction of argininosuccinate synthetase by cytokines.

Nitric oxide (NO) may be a mediator of beta-cell damage in insulin-dependent diabetes mellitus. beta-Cells express the inducible form of NO synthase (iNOS) and produce large amounts of NO upon exposure to cytokines. iNOS requires the amino acid arginine for NO formation. It has been shown in other cell types that interferon-gamma (IFN gamma) and bacterial lipopolysaccharide induce the enzyme argininosuccinate synthetase (AS), enhancing the capacity of these cells to regenerate arginine from citrulline and maintain NO production in the presence of low arginine concentrations. To characterize the messenger RNA (mRNA) expression of AS in insulin-producing cells, RINm5F cells (RIN cells) were exposed to interleukin-1 beta (IL-1 beta) or to tumor necrosis factor-alpha plus IFN gamma. After 4-6 h, there was a significant and parallel induction of AS and iNOS mRNA. IL-1 beta-induced AS and iNOS mRNA expression was prevented by an inhibitor of the activation factor NF-kappa B pyrrolidine diaminoguanidine, an inhibitor of gene transcription (actinomycin D), and a blocker of protein synthesis (cycloheximide), suggesting coregulation of AS and iNOS by cytokines. RIN cells exposed to IL-1 beta in the presence of citrulline but the absence of arginine had increased AS enzyme activity and produced NO, demonstrating that cytokine-induced AS mRNA expression is accompanied by increased AS activity. Both adult rat islets exposed to IL-1 beta and human pancreatic islets cultured in the presence of IL-1 beta, tumor necrosis factor-alpha, and IFN gamma were able to use citrulline to regenerate arginine and produce NO. Taken as a whole, the present data suggest that regulation of AS activity may play a role in modulation of NO production in both rodent and human insulin-producing cells.

Pyrrolidine dithiocarbamate prevents IL-1-induced nitric oxide synthase mRNA, but not superoxide dismutase mRNA, in insulin producing cells.

We presently investigated the effects of pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of nuclear factor kappa B (NF-kappa B), on the induction of nitric oxide synthase (iNOS) and manganese superoxide dismutase (MnSOD) mRNAs by IL-1 beta in insulin-producing RIN cells. PDTC decreased by 90% both IL-1 beta-induced increase in medium nitrite accumulation (an indicator of NO production) and induction of iNOS mRNA expression. However, PDTC did not prevent induction of MnSOD mRNA by IL-1 beta. PDTC induced an early (45 min) expression of c-fos mRNA and potentiated IL-1 beta-induced c-fos expression. Our data suggest that NF-kappa B activation is necessary for iNOS, but not for MnSOD, mRNA expression in insulin producing cells.